The major thrust of the proposed work is to develop fluorescent indicator systems for free zinc in the brain, with improved selectivity, sensitivity, and calibratable response both intracellulady and extracellularly. Current small molecule indicators are insufficiently sensitive, selective, or both, and many do not respond in a fashion (intensity ratio, anisotropy, or lifetime) that can be accurately calibrated in situ. Our efforts have already met some success, and we anticipate that they will be valuable in understanding the physiological roles of zinc in the brain in vivo, as well as its pathological roles in ischemic stroke, epilepsy, Alzheimer's disease, and blunt trauma. Our specific aims are focused on developing new methods and using them to answer questions of importance in zinc neurobiology. They include quantitatively imaging free zinc levels in the cell nucleus, using an excitation ratiometric approach; measuring zinc release in a dog ischemia model using a novel fiber optic sensor;, measuring zinc levels during seizures in a rat epilepsy model; and quantitatively imaging zinc levels in mitochondria. If successful, we anticipate that our technology will be of use in understanding and developing treatments for the conditions listed above.